Fluorescence Imaging
Currently, magnetic resonance (MR), ultrasonic (US) and computer tomographic (CT) imaging techniques are major tools for clinical diagnosis of diseases and evaluation of therapeutic interventions. Microscopic imaging techniques, for instance, those based on fluorescent dyes constitute an effective and complementary approach for acquiring microstructural information that can be used to discriminate among tissue types, study the progression of diseases in tissue and cells, and evaluate potential treatment options for such diseases.
Fluorescence microscopy is an indispensable tool in cell biology because the fluorescence labelling of proteins, molecules and spaces enables the study of structures and functions in biologic specimens. Typically, fluorescence microscopy has not been used to examine tissue in situ because of the need for close association between microscope instrumentation and the imaged tissue, toxic or expensive fluorescent dyes for image contrast, and relatively long image acquisition times. Despite these challenges, fluorescence microscopy techniques have been shown to provide valuable diagnostic information for various disease states. Studies with biopsy specimens suggest that fluorescence imaging can provide useful diagnostic information about the presence of precancerous lesions; confocal images of normal and dysplastic cervical biopsy specimens obtained with a confocal reflectance microscope showed a strong correlation between nuclear morphologic features extracted from fluorescence images and histopathologic diagnosis.
Fluorescent microscopy techniques include confocal microscopy that allows creation of high resolution images and differ from conventional optical microscopy in that they use a condenser lens to focus illuminating light of specific wavelengths from a light source, e.g., a laser, into a very small, diffraction limited spot within a specimen, and an objective lens to focus the light emitted from that spot onto a small pinhole in an opaque screen. A detector, which is capable of quantifying the intensity of the light that passes through the pinhole at any instant, is located behind the screen. Because only light from within the illuminated spot is properly focused to pass through the pinhole and reach the detector, any stray light from structures above, below, or to the side of the illuminated spot are filtered out. The image resolution is therefore greatly enhanced as compared to other conventional tissue imaging approaches.
In a scanning confocal microscopic imaging system, a coherent image is built up by scanning point by point over the desired field of view and recording the intensity of the light emitted from each spot, as small spots are illuminated at any one time. Scanning can be accomplished in several ways, including for example and without limitation, via laser scanning. Confocal microscopic imaging system are commercially available through entities such as Carl Zeiss, Nikon, Leica, and Olympus, including, for example and without limitation, a Zeiss LSM 5 Duo, a Leica FCM1000, and the like. An exemplary confocal microscopic imaging system is described in U.S. Pat. No. 6,522,444 entitled “Integrated Angled-Dual-Axis Confocal Scanning Endoscopes,” which is assigned to Optical Biopsy Technologies, Inc.
The ability to obtain fluorescence images of normal and diseased tissue in situ is limited by the ability to bring the tissue of interest in close proximity to the objective lens of the microscope. Fluorescence microscopic imaging systems incorporating either a solitary optical fiber or a fiber optic imaging bundle are needed to facilitate in situ imaging of less accessible organ sites. Similarly, miniaturized fluorescent microscopic systems allow for imaging of organs and tissues in situ. However, a major obstacle for application of fluorescence microscopic imaging techniques is related to the introduction of fluorescent dyes into biological tissue. Commonly, introduction of dye is performed by infusion or systemic needle injection. Disadvantages of these methods include, for example, the high dosages of the dye(s) required for imaging, wash-out (release of the dye(s) by the tissue), and inhomogeneous distribution of the fluorescent dye.
Imaging of Cardiac Tissue
Quantity, density, and morphology of cardiac cells vary significantly during development, amongst species, for each cardiac tissue and in heart disease. Many diseases, such as hypertrophy, atrophy, infarction, and ischemia, are known to be associated with alterations in cell geometry and density. For instance, in cardiac hypertrophy, human epicardial left ventricular myocytes have been shown to increase in length, width, area, and volume by approximately 9%, 28%, 39%, and 78%, respectively, and rabbit right ventricular myocytes are known to increase in length and width by approximately 7.5% and 36%, respectively. In atrophic hearts, left ventricular myocytes decrease in volume by 50%-75%, with little change in myocyte length. Cardiac diseases are also known to alter the extracellular environment. Following myocardial infarction, fibrosis (excessive deposition of extracellular matrix mediated by fibroblasts) occurs not only in the infarcted region, but in the surrounding regions as well. Furthermore, early stages of ischemia are known to decrease the extracellular resistance, which is indicative of reduced interstitial space.
A more comprehensive understanding of these pathologic cellular and tissue alterations could allow the recently developed fiber-optics confocal systems and optical imaging techniques to provide a new set of diagnostic tools in cardiology.
In previous studies, pathologic alterations of cardiac microstructure have been characterized ex vivo with confocal microscopy. However, the application of confocal microscopy requires that fluorescent dye for labeling of proteins or structures is available in sufficient concentration in the region of interest. Dye delivery is commonly a time-consuming immunochemistry procedure, requiring excision, fixation, and sectioning of tissue as well as cell membrane disruption. In particular, in vivo dye delivery is an unresolved issue that impedes the application of fiber-optics confocal imaging in these studies.
Image data from both living and fixed tissue specimens have been used to develop models that describe physical and physiological properties of cardiac tissue. For instance, models that describe mechanical and electrophysiological properties in normal and diseased cells and tissues have been developed. Most of these models do not directly account for the detailed tissue microstructure, but describe tissue properties with lumped parameters or homogenization approaches. A small number of models have been introduced, which are based on an analytical description of microstructure or on two-dimensional microscopic images.
Cardiac tissue can be viewed as a composite material comprised of fluids and cells, including myocytes, fibroblasts, endothelial, vascular smooth muscle, and neuronal cells. Myocytes occupy most of the volume in cardiac tissue and are responsible for cardiac contraction. The (interstitial) space between cardiac cells is filled with fluid and an interconnected extracellular matrix comprised mostly of collagen and capillary vessels.
Myocytes in ventricular and atrial tissue exhibit a micro-structural organization that underlies physical and physiological properties, such as electrical conductivity and electrical wave velocity, respectively. Other components of the heart have differing micro-structural arrangements, such as the strand like fibers of the Purkinje system.
There are several complications associated with heart surgery, including dysfunction of sino-atrial and atrio-ventricular conduction pathways. These complications require chronic cardiac rhythm management using implantable pacemakers. Despite the complexity and individual variations in cardiac conductive pathways, tissue discrimination during surgery is currently limited to the use of anatomic landmarks, and accurate surgical intervention is challenging and risky. One of the perioperative complications that can be induced during these procedures is complete heart block that is purportedly associated with interruption of cardiac conduction pathways.
Therefore, what is needed in the art are fluorescence imaging systems and methods that achieve in vivo imaging and microstructural characterization of tissues while avoiding high dosages of fluorescent dye(s), undesired wash-out, and inhomogeneous distribution of fluorescent dye(s) within the tissue. There is a further need in the art for systems and methods of producing detailed images of tissue microstructure in real time during the performance of surgical procedures. There is still a further need in the art for systems and methods of identifying conductive pathways within a tissue to avoid damage to such conductive pathways during a surgical procedure.